Liposomes with a negative excess charge

ABSTRACT

The present invention concerns liposomes with a negative excess charge and pharmaceutical compositions produced therefrom. The liposomes contain 30 to 50 mol % cholesterol, 49 to 56 mol % phospholipids and 1 to 14 mol % of one or several compounds of the general formula I ##STR1## or salts thereof in relation to the total lipid components of the liposomes, in which R 1  and R 2  are the same or different and represent hydrogen, C 1  -C 4  alkyl groups or saturated or unsaturated C 8  -C 24  acyl groups which are unbranched or branched or/and unsubstituted or substituted, provided that at least one of the residues R 1  and R 2  is an acyl group as defined above. A process for the production of the liposomes in accordance with the invention is also provided.

DESCRIPTION

The invention concerns liposomes with a negative excess charge, inparticular for administering cytostatic agents and cytokines.

Liposomes are spherical structures comprising one or several lipiddouble layers with an aqueous inner space (lipid vesicle). Such vesiclescan be produced by the mechanical very fine dispersion of phospholipids(e.g. lecithin) in aqueous media.

Bangham et al., J. Mol. Biol. 13 (1965), 238-252 observed thatphospholipids form superstructures in the presence of water. Dependingon physical parameters such as pressure, temperature and ionicconcentration, micelles, unilamellar or multilamellar liposomes or evensimple lipid double layers form (cf. Liposomes: From physical structureto therapeutic application (1981), Knight, C. G. (ed.), Elsevier, NorthHolland Biomedical Press, chapter 2: H. Eibl, Phospholipid synthesis,19-50; chapter 3: F. Szoka and D. Papahadjopoulos, Liposomes:Preparation and Characterization, 51-104). Small unilamellar liposomesare spherical structures with a diameter of 20 to 200 nm (cf. Barenholtzet al., FEBS Let. 99 (1979) 210-214). Their inner volume is comprised ofan aqueous medium which is bordered on the outside by the lipid doublelayer. Depending on their lipophilicity or hydrophilicity, activesubstances can either be entrapped in the lipid double layer or in theaqueous inner volume of the liposomes and transported and distributed inthe organism via the body fluids.

Due to their structure, liposomes serve as a model for membranes inbiochemistry and molecular biology. In past years numerous papers on theproperties of liposomes and their use as carriers of medicinal agentshave also been published (cf. e.g. H. Schreiner and M. Raeder-Schikorr,"Pharmazie in unserer Zeit" 11 (1982), 97-108). Previously publishedexperiments on animals generally show that the liver and spleen dominateover other organs with respect to the uptake of liposomes. About 8% ofthe liposomes are found in the liver after 1 hour and about 15% after 24hours.

The possible use of liposomes in medicine is mainly aimed at theselective treatment of diseases. The desired effects of the activesubstance entrapped in the liposomes should be promoted whereas theundesired effects should be reduced. In this manner it is intended toachieve an improvement in the therapeutic index.

Liposomes are known from DE-OS 40 13 632.9 which contain at least 1 mol% of a compound having a positive excess charge.

However, a disadvantage of administering known liposomes is that theiruptake in the liver is relatively limited and that the liposomes cancirculate for a long period in the blood. This applies particularly toliposomes which are composed of phospholipids such as lecithin andcholesterol. In this manner the active substance contained in theliposomes is distributed throughout the body which in turn can lead toan increase in the occurrence of undesired side effects of the activesubstance.

An object of the present invention was therefore to provide newliposomes which exhibit an increased liver uptake compared to liposomesof the state of the art.

This object is achieved by the provision of liposomes which arecharacterized in that they contain cholesterol, phospholipids and 1 to14 mol % relative to the total lipid components of the liposomes of oneor several compounds of the general formula I ##STR2## or their salts,in which R¹ and R² can be the same or different and represent hydrogen,C₁ -C₄ alkyl groups or saturated or unsaturated C₈ -C₂₄ acyl groupswhich can if desired, be branched or/and substituted provided that atleast one of the residues R¹ and R² is an acyl group as defined above.

Under physiological conditions the above compounds (I) are generally inthe form of partially protonated anions so that the liposomes accordingto the invention have a negative excess charge. The amount of negativelycharged carrier substances according to formula (I) is generally 1 to 14mol %, preferably 3 to 10 mol % relative to the total lipid componentsof the liposomes. An increase in the amount of compounds of formula (I)beyond 15% leads to solubility problems which can go as far asflocculation. Therefore the preferred range is usually adhered to.

Those compounds of formula (I) are preferred in which either bothresidues represent C₈ -C₂₄ acyl groups or one residue representshydrogen and the other residue represents a C₈ -C₂₄ acyl group.

Those compounds are particularly preferred in which R¹ is a C₁₄ -C₂₄acyl group and R² is hydrogen. Preferred examples from this group are inturn 1-stearoyl-sn-glycero-3-phosphoric acid,1-palmitoyl-sn-glycero-3-phosphoric acid,1-myristoyl-sn-glycero-3-phosphoric acid and1-erucoyl-sn-glycero-3-phosphoric acid as well as salts thereof. Thesephosphatidic acids are preferably used in the form of salts e.g. in theform of monoalkali metal salts, in particular monosodium salts.

In a further particularly preferred class of compounds R¹ and R² are C₁₄-C₂₄ acyl groups. Preferred examples from this class are in turn1,2-distearoyl-sn-glycero-3-phosphoric acid,1,2-dipalmitoyl-sn-glycero-3-phosphoric acid,1,2-dimyristoyl-sn-glycero-3-phosphoric acid and1,2-dierucoyl-sn-glycero-3-phosphoric acid as well as salts thereof.These phosphatidic acids are also preferably used in the form of theirsalts e.g. in the form of their monoalkali metal salts, in particular asmonosodium salts.

Surprisingly it was found that a liposome which contains a negativelycharged carrier substance according to formula (I) has considerableadvantages compared to known liposomes that have been prepared withoutusing compounds according to formula (I).

When liposomes of the state of the art were administered, it was namelyfound that their uptake in the liver is relatively limited and that theytherefore circulate for quite a long period in the blood. In contrast asubstantially improved liver uptake was surprisingly found for liposomesaccording to the invention which in turn leads to a major decrease inthe side effects in other organs of the pharmaceutical active substancesentrapped in the liposomes.

This was investigated in particular for the anthracyclin antibioticdoxorubicin. This substance has already been used clinically for about25 years. However, its use as an anti-tumour agent in the treatment ofsolid tumours, leukaemias and lymphomas (Bonadonna et al., Cancer Res.30 (1970), 2572-2582; Middleman et al., Cancer 28 (1971), 844-850; Tanet al., Cancer 32 (1973), 9-17) was very limited due to its high cardiactoxicity (Lefrak et al., Cancer 32 (1973), 302-314; Rinehart et al.,Ann. Internal. Med. 81 (1974), 475-478; von Hoff et al., Ann. Internal.Med. 91 (1979), 710-717).

In the studies which led to the present invention it was found byexperiments on mice that liposomes according to the invention whichcontain doxorubicin as the active substance have on the one hand acomparable plasma stability to known liposomes but, on the other hand,surprisingly differ greatly from these with regard to pharmacokinetics.When using liposomes according to the invention, considerably smalleramounts of doxorubicin were found in the plasma, in the lung, in thekidney and in particular also in the heart. In contrast in the liver,the desired target organ of the liposomes, a considerably higherconcentration of doxorubicin was found when using liposomes according tothe invention than when using liposomes of the state of the art or thanwhen using free doxorubicin.

A further surprising advantage of the liposomes according to theinvention is that the metabolism of the active substance containedtherein differs from the metabolism of an active substance administeredwithout liposomes. Thus when doxorubicin is administered in the form ofa commercial preparation (without liposomes), after 1 hour only 20% ofthe doxorubicin remains in the liver while the rest has already beenmetabolized. In contrast the administration of the same active substancein the liposomes according to the invention results in 97% of the activesubstance in a non-metabolized form after 1 hour under otherwise thesame conditions so that in this case a metabolism of only 3% occurs. Incontrast in the case of commercial preparations (without liposomes) ca.80% of the active substance has already been metabolized in the liverafter 1 hour.

The liposomes according to the invention contain cholesterol andphospholipids as further lipid components in addition to compounds ofthe general formula (I). The liposomes preferably contain 30 to 50 mol %cholesterol and 49 to 56 mol % phospholipids. Uncharged phospholipidsare particularly preferred. The term "uncharged phospholipids" is to beunderstood as those phospholipids which are uncharged externally (i.e.the term includes those phospholipids which are intramolecularzwitterions). A preferred class of phospholipids in the liposomesaccording to the invention are phosphoglycerides i.e. compounds thatcontain a glycerol group in which 2 hydroxyl groups of the glycerol areesterified by fatty acid groups (preferably C₈ -C₂₄ acyl groups) and thethird hydroxyl group is esterified by a phosphorylated alcohol. Examplesof particularly suitable alcohols are those which carry a positivecharge such as ethanolamine and choline. Phospholipids are particularlypreferred which are lecithins of the general formula (II) ##STR3## inwhich R³ and R⁴ can be the same or different and represent saturated orunsaturated C₈ -C₂₄ acyl groups which if desired, can be branched or/andsubstituted. R³ and R⁴ are preferably C₁₄ -C₂₄ acyl groups. R³ and R⁴are particularly preferably stearoyl, palmitoyl, myristoyl or erucoylresidues.

The components of the liposomes according to the invention are eithercommercially available or can be prepared by known processes. Lecithinsof formula (II) can for example be prepared according to Woolley andEibl, Chem. Phys. Lipids 47 (1988), 55-62 and Eibl and Woolley, Chem.Phys. Lipids 41 (1986), 53-63. Furthermore the production ofphosphoglycerides is described in DE-OS 32 25 213.7 and the literaturereferences cited therein. Compounds of the general formula (I) can forexample be prepared from the respective lecithins by cleaving off thecholine residue with phospholipase D (Eibl and Woolley, Methods Enzymol.72 (1981), 632-639).

In order to produce the liposomes according to the invention, theindividual lipid components of the liposomes i.e. 1 to 14 mol % ofcompounds of the general formula (I) and the other lipid components inan amount which together with compounds of formula (I) amounts to 100mol %, are dissolved in a suitable solvent, preferably while heating, inorder to achieve a homogeneous mixing of the components. The solvent isremoved in a vacuum and the finely dispersed lipid film is admixed withan aqueous buffer solution (all solutions that can be usedphysiologically can be used as the aqueous buffer solution).Subsequently the mixture is kept for ca. 1 hour while stirring gently ata temperature which is generally about 5° C. above the main transitiontemperature of the lipids e.g. at 50° C.

This pre-heated lipid suspension is then converted by suitable measuresin a known manner e.g. in a pressure cell of a French press (AmincoCompany, Silverspring, USA) into the liposomes. The French press iscomposed of a hydraulic press and a standard pressure cell made ofsteel. After closing the pressure cell, the pressure is increased andthe resulting liposome dispersion is pressed under constant pressurethrough as small outlet. The process is repeated at least three times.After centrifuging the liposome dispersion, the supernatant is removedfrom the sediment. It contains the liposomes that are now available forvarious applications and investigations e.g. for the production ofliposomes which contain one or several pharmaceutical active substances.The liposomes according to the invention can, however, also be producedby other methods.

The present invention therefore concerns a process for the production ofliposomes in which 1 to 14 mol % of compounds of the general formula (I)together with the other lipid components of the liposomes in an amountwhich together with the compounds of the general formula (I) amounts to100 mol %, are converted into a lipid suspension, the lipid suspensionis pre-heated and the lipid suspension pre-heated in this manner is thenconverted in a known manner by suitable measures into liposomes.

The present invention in addition concerns a pharmaceutical preparationcontaining the liposomes according to the invention and one or severalpharmaceutical active substances entrapped in the liposomes, if desired,together with common pharmaceutical diluents, auxiliary substances,carrier substances and fillers.

All active substances which can in general be introduced into plasma bymeans of liposomes can be used as the active substances. Preferredgroups of active substances are on the one hand cytostatic agents, inparticular anthracyclin antibiotics such as doxorubicin, epirubicin ordaunomycin, doxorubicin being particularly preferred. Further preferredcytostatic agents are idarubicin, hexadecylphosphocholine,1-octadecyl-2-methyl-rac-glycero-3-phosphocholine, 5-fluoruracil,cis-platinum complexes such as carboplatin and novantron.

Further preferred groups of active substances are immunomodulatingsubstances such as cytokines, among these the interferones and inparticular α-interferon being particularly preferred, antimycoticallyactive substances (e.g. amphotericin B) and substances active againstprotozoal diseases (malaria, trypanosomal and Leishmania infections.Taxol is also preferred as an active substance.

In addition the present invention therefore concerns the use ofliposomes according to the invention to produce an anti-tumour agent, inwhich case the active substance is particularly preferably doxorubicin.

The present invention in addition concerns the use of the liposomesaccording to the invention to produce an agent to influence cellproliferation in which case the active substance is a cytokine,particularly preferably α-interferon.

In order to produce a pharmaceutical composition according to theinvention which contains one or several active substances that areentrapped in the liposomes according to the invention, the procedure isas follows:

In order to entrap water-insoluble substances, the active substance isdissolved together with the lipids in a suitable solvent such asmethylene chloride or chloroform, subsequently the liposomes areproduced according to the process described above.

In order to entrap water-soluble substances, the lipid film is admixedwith a solution as described above which, however, now contains thewater-soluble active substance. Subsequently, the further procedure isas described above. The supernatant after centrifugation also containsthe unentrapped water-soluble active substance in addition to the filledliposomes. This free portion of active substance can be separated fromthe portion enclosed in the liposomes by for example diafiltration.Before using the liposomes it is preferable to in addition carry outsterile filtration with membrane filters (Sartorius Company, porediameters 0.2 μm).

The present invention therefore also concerns a process for theproduction of a pharmaceutical composition in which the processdescribed above is used to produce liposomes and, in order to entrapwater-insoluble active substances, the active substance is dissolvedtogether with the lipid components and, in order to entrap water-solubleactive substances, the lipid film is admixed with an aqueous solutionthat contains the water-soluble active substance.

In the following the invention is further elucidated in the examples.

EXAMPLE 1

Production of liposomes

1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was obtained fromSygena LTD (Liestal, CH) and cholesterol (Ch) was obtained from Fluka(Buchs, CH). 1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) wasprepared via 1,2-distearoyl-sn-glycerol (Eibl and Woolley, Chem. Phys.Lipids 41 (1986), 53-63). 1,2-distearoyl-sn-glycero-3-phosphate (DSPA)was produced by hydrolysis of 1,2-distearoyl-sn-glycero-3-phosphocholineusing phospholipase D (Eibl and Woolley, Methods Enzymol. 72 (1981),632-639). Hydrogenated soya phosphatidylinositol (HPI) was prepared fromsoya lecithin (P5638) that was obtained from "Sigma Chemie GmbH(Steinheim, Germany)". Soya lecithin contains about 10%phosphatidylinositol which was isolated by silica gel chromatography(Woolley and Eibl, Chem. Phys. Lipids 47 (1988), 55-62). The resultingproduct was hydrogenated in 100 ml CHCl₃ /CH₃ OH (1:1, v/v) with 10%Pd/C catalyst.

Small unilamellar liposomes having the following compositions (on amolar basis) were produced: DPPC/Ch (6:4); DSPS/DSPA/Ch (5:1:4);DPPC/HPI/Ch (5:1:4). For this 5 mmol of the lipid suspensions weremechanically dispersed in 50 ml citric acid buffer (300 mmol/l, pH 4)for 3 hours in a rotary evaporator at temperatures 5° C. above the phasetransition temperature (T_(M)) of the phospholipid with the highestT_(M). Liposomes were produced from this homogeneous solution using aFrench press at 20,000 psi. The resulting liposomes were centrifuged for30 minutes at 30,000 g. 100 mg doxorubicin in powder form was added tothis and entrapped at pH 8 using the loading procedure of Mayer et al.(Biochim. Biophys. Acta 857 (1986), 123-126). Free doxorubicin that wasnot entrapped in the liposomes was removed by diafiltration (PolysulfoneUltrasart Module, exclusion limit 20 kD, Sartocon Mini SM 17521,Sartorius, Germany). All liposomes were sterilized by filtration beforeuse.

EXAMPLE 2

Treatment of mice with liposomes containing doxorubicin

Female NMR-I mice (28-32 g, 6-8 weeks) were obtained from the AnimalBreeding Institute, Hannover Germany. The same doses (5.7 mg/kg) wereinjected in each case into the cordal vein of the mice. Each treatmentgroup was composed of 3 animals. After 1, 24 and 72 hours the mice wereanaesthetized with CO₂. Blood was collected from the mice by puncture ofthe heart and stored in heparinized Eppendorf vessels. Tissue samplesfrom the mice (liver, heart etc.) were rinsed in 0.9% NaCl solution andstored at -70° C.

The plasma samples were analyzed by the procedure of Mross et al., (J.Chromatograph. 530 (1990), 192-199). For this 100 μl plasma was loadedonto activated Bond Elut C18 columns (ICT, Frankfurt, Germany) andrinsed with 4 ml buffer (0.02 mol/l Na phosphate, pH 3.9). The columnswere dried for 10 minutes at 10 mbar. Doxorubicin and its metaboliteswere eluted with 4 ml methanol/chloroform (1:1, v/v). The eluate wasevaporated to dryness under nitrogen. The residue was taken up in 50 μl(0.02 mol/l Na phosphate/acetonitrile, pH 3.9; 7:3, v/v) and analyzed bymeans of HPLC using a 4.6 mm×25 cm Merck Lichrocart C 18 RP 5 μm column.The flow rate was 1.5 ml/min. The excitation wavelength was 480 nm andthe emission wavelength was 580 nm.

In order to detect doxorubicin and its metabolites in the individualorgans, the tissue samples (about 200 mg in each case) were homogenizedwith a micro-dismembrator (Braun Melsungen AG, Germany). Doxorubicin andits metabolites were treated with an extraction solution at pH 3 (16.5%w/v AgNO₃ in acetonitrile/water; 6:4, v/v), in order to release theanthracyclines from a complex with DNA and to precipitate the proteins.After addition of an internal standard (epirubicin) and centrifugation,the supernatant was applied directly to the HPLC column.

The DPPC/Ch liposomes had an average diameter of 64 nm, the DPPC/HPI/Chliposomes had an average diameter of 91 nm and the DSPC/DSPA/Chliposomes according to the invention had an average diameter of 88 nm.

The average ratio of doxorubicin to phospholipid was 170 μg/μmol forDPPC/Ch liposomes (95% entrapment), 78 μg/μmol for DSPC/DSPA/Chliposomes (65% entrapment) and 65 μg/μmol for DPPC/HPI/Ch liposomes (71%entrapment). The three liposome types were stable in plasma for at least24 hours. Even after 3 months storage at 4° C. no release of doxorubicinfrom the liposomes could be found.

The dependence of the pharmacokinetics of doxorubicin on the type offormulation is given by the following results: after 1 hour the plasmavalue was 0.5 μg/ml when free doxorubicin (without liposomes) was addedand only traces of doxorubicin could be found after 24 hours. TheDPPC/Ch and DPPC/HPI/Ch liposomes exhibited a very high plasma levelafter 1 hour (59.3 and 42.9 μg/ml respectively) and also exhibited arelatively high level up to 24 hours (5.5 and 4.9 μg/ml respectively).In contrast, only 7.7 μg/ml doxorubicin could be detected in plasmaafter 1 hour with the DSPC/DSPA/Ch liposomes according to the invention.After 24 hours only traces of doxorubicin could be found.

In the heart the level of free doxorubicin was 13 μg/g after 1 hourwhereas with the DSPC/DSPA/Ch liposomes according to the invention only1.0 μg/g was found. The corresponding values for DPPC/Ch and DPPC/HPI/Chliposomes were about 3.5 μg/g heart tissue. In the lung and in thekidney considerably lower concentrations of the active substance werealso found after 1 hour when doxorubicin was administered inDPPC/DSPA/Ch liposomes compared to the other forms of administration.

Differences in the doxorubicin concentration depending on the form ofadministration were also found in the liver. After 1 hour thecorresponding doxorubicin values were as follows: 11.0 μg/g for freedoxorubicin, 42.8 μg/g for the DSPC/DSPA/Ch liposomes according to theinvention, 30 μg/g for DPPC/HPI/Ch liposomes and 12.2 μg/g for DPPC/Chliposomes. The liposomes according to the invention also showed a highvalue of 25.1 μg/g doxorubicin in the liver even after 24 hours. Evenafter 72 hours the concentration of doxorubicin in this tissue was stillremarkably high.

The following was found concerning the metabolism of doxorubicin in theliver: when free doxorubicin was administered 23% active agent comparedto 77% inactive metabolites (aglycones) was found after 1 hour. Incontrast, 97% active doxorubicin compared to 3% inactive aglycones wasobserved for the DSPC/DSPA/Ch liposomes according to the invention. Thecorresponding values were 96%:4% for DPPC/Ch liposomes and 90%:10% forDPPC/CH liposomes.

As in the liver, an increased uptake of doxorubicin was found in thespleen after 1 hour when using DPPC/DSPA/Ch liposomes.

In summary it was established that the administration of doxorubicin inliposomes according to the invention is superior to the administrationof the active substance in a free form and also to the administration ofthe active substance in other liposomes in particular due to the highertissue specificity.

What is claimed is:
 1. Unilamellar liposomes containing a pharmaceuticalactive substance,wherein the liposomes contain 30 to 50 mol %cholesterol, 49 to 56 mol % of at least one first phospholipid and 1 to14 mol % of at least one second phospholipid of the general formula I##STR4## or salts thereof in relation to the total lipid components ofthe liposomes, in which R¹ and R² are the same or different andrepresent hydrogen, C₁ -C₄ alkyl groups or saturated or unsaturated C₈-C₂₄ acyl groups which are unbranched or branched or/and unsubstitutedor substituted, provided that at least one of the residues R¹ and R² isan acyl group as defined above, wherein the liposomes are filteredthrough a filter having a pore diameter of 0.2 μm.
 2. Liposomes asclaimed in claim 1 whereinthe liposomes contain lecithins of the generalformula II as the at least one first phospholipid ##STR5## in which R³and R⁴ can be the same or different and represent saturated orunsaturated C₈ -C₂₄ acyl groups which are unbranched or branched or/andsubstituted or unsubstituted.
 3. Liposomes as claimed in claim 2,whereinR³ and R⁴ are C₁₄ -C₂₄ acyl groups.
 4. Liposomes as claimed in claim3,wherein R³ and R⁴ are selected from the group consisting of stearoyl,palmitoyl, myristoyl and erucoyl residues.
 5. Liposomes as claimed inclaim 1, wherein the at least one second phospholipid isdistearoylphosphatidic acid and the at least one first phospholipid isdistearoylphosphocholine.
 6. Liposomes as claimed in claim 1,wherein theliposomes contain 3 to 10 mol % of at least one second phospholipid offormula (I) or a salt thereof.
 7. Liposomes as claimed in claim1,wherein R¹ is a C₁₄ -C₂₄ acyl group and R² is hydrogen.
 8. Liposomesas claimed in claim 7,wherein, the at least one second phospholipid offormula (I) is selected from the group consisting of1-stearoyl-sn-glycero-3-phosphoric acid,1-palmitoyl-sn-glycero-3-phosphoric acid,1-myristoyl-sn-glycero-3-phosphoric acid and1-erucoyl-sn-glycero-3-phosphoric acid as well as salts thereof. 9.Liposomes as claimed in claim 8,wherein, the at least one secondphospholipid is a monosodium salt of the said acids.
 10. Liposomes asclaimed in claim 1,wherein, R¹ and R² are C₁₄ -C₂₄ acyl groups. 11.Liposomes as claimed in claim 10,wherein, the at least one secondphospholipid is selected from the group consisting of1,2-distearoyl-sn-glycero-3-phosphoric acid,1,2-dipalmitoyl-sn-glycero-3-phosphoric acid,1,2-dimyristoyl-sn-glycero-3-phosphoric acid and1,2-dierucoyl-sn-glycero-3-phosphoric acid as well as the salts thereof.12. Liposomes as claimed in claim 11,wherein, the at least one secondphospholipid is a monosodium salt of the said acids.
 13. Liposomes asclaimed in claim 1,wherein, the at least one first phospholipid is aphosphoglyceride.
 14. Liposomes as claimed in claim 1,wherein, theliposomes have a diameter of less than 100 nm.
 15. Liposomes as claimedin claim 1, wherein the active substance is selected from the groupdoxorubicin, anthracyclin antibiotic, idarubicin, carboplatin, taxol andcytokine.
 16. A process for the production of unilamellar liposomescontaining a pharmaceutical active substance, wherein the liposomescontain 30 to 50 mol % cholesterol, 49 to 56 mol % of at least one firstphospholipid and 1 to 14 mol % of at least one second phospholipid ofthe general formula I ##STR6## or salts thereof in relation to the totallipid components of the liposomes, in which R¹ and R² are the same ordifferent and represent hydrogen, C₁ -C₄ alkyl groups or saturated orunsaturated C₈ -C₂₄ acyl groups which are unbranched or branched or/andunsubstituted or substituted, provided that at least one of the residuesR¹ and R₂ is an acyl group as defined above, the process comprising:(a)dissolving the at least one second phospholipid of the general formulaI, the cholesterol and the at least one first phospholipid in a solvent,wherein the at least one first phospholipid of general formula I, thecholesterol and the at least one second phospholipid amount to 100 mol%, (b) removing the solvent in a vacuum to produce a finely dispersedlipid film, (c) mixing the finely dispersed lipid film with an aqueousbuffer solution to form a lipid suspension, wherein the aqueous buffersolution comprises the pharmaceutical active substance, (d) heating thelipid suspension, and (e) forming liposomes from the lipid suspension ofstep (d) whereby liposomes containing the pharmaceutical activesubstance are produced, and thereafter filtering the liposomes through afilter having a pore diameter of 0.2 μm.
 17. The process as claimed inclaim 16, wherein the pharmaceutical active substance is water-soluble.18. The process as claimed in claim 16, further comprising:(f)separating the liposomes of step (e) from the lipid suspension of step(e).
 19. A process for the production of unilamellar liposomescontaining a water-insoluble pharmaceutical active substance, whereinthe liposomes contain 30 to 50 mol % cholesterol, 49 to 56 mol % of atleast one first phospholipid and 1 to 14 mol % of at least one secondphospholipid of the general formula I ##STR7## or salts thereof inrelation to the total lipid components of the liposomes, in which R¹ andR² are the same or different and represent hydrogen, C₁ -C₄ alkyl groupsor saturated or unsaturated C₈ -C₂₄ acyl groups which are unbranched orbranched or/and unsubstituted or substituted, provided that at least oneof the residues R¹ and R² is an acyl group as defined above, the processcomprising:(a) dissolving the at least one of second phospholipid of thegeneral formula I, the cholesterol, the at least one first phospholipidand the water-insoluble active substance in a solvent, wherein the atleast one second phospholipid of general formula I, the cholesterol andthe at least one first phospholipid amount to 100 mol %, (b) removingthe solvent in a vacuum to produce a finely dispersed lipid film, (c)mixing the finely dispersed lipid film with an aqueous buffer solutionto form a lipid suspension, (d) heating the lipid suspension, and (e)forming liposomes from the lipids suspension of step (d) wherebyliposomes containing the water-insoluble active substance are produced,and thereafter filtering the liposomes through a filter having a porediameter of 0.2 μm.